VP 177AD-1 Experimental Data 

Abstract

The VP 177AD-1 is a 96-channel dispensing manifold designed for rapid filling of both standard and deep 96-well.  This experiment was setup to determine both the accuracy and precision of this instrument by measuring the quantity of aqueous FITC dispensed per well and the coefficient of variance from well to well.  A plate filled by a 12-channel pipette was used as a standard control to calculate how much FITC is transferred by the manifold.  The manifold was tested in volumes of 50, 100, and 200 uL dispenses.  CV’s for these transfers ranged from 3.5% to 8.2%, and the volume transferred were within 3.1% of the target dispense volume.  CV’s for the multi-channel pipettor used for the standard curve ranged from 4.6% to 12.5%.  The experiment supports the conclusion that the VP 177AD-1 can rapidly fill 96-well plates compared to an 8 or 12-channel pipettor while maintaining equivalent reliability.

  I.  Overview

BACKGROUND INFORMATION

The VP 177AD-1 is a dispensing manifold designed for rapid filling of 96-well plates.  It can dispense volumes ranging from 50-500 uL per well, and has Y and Z-axis adjustability.  Plates can be filled with reagents by sliding plates under the manifold, adjusting the Z-Height, dispensing, and reposition the tubes to the side of the wells to wick off hanging drops as the manifold is raised.  Volume transferred can be adjusted with a VP 195K (or 195D-1) bottle-top dispenser.

OBJECTIVE

The purpose of this assay was to determine the accuracy and precision of the VP 177AD-1 manifold dispensing into a 96-well microplate.  Accuracy was measured by the percent of volume actually transferred to target volume and precision by the coefficient of variance of this transfer from well to well.  Transfers of 50, 100, and 200 uL per well were tested.

FITC Dispense Assay Summary

• M Tris –HCl, pH 8.0, was added to 96-well Black Assay Plates (Greiner Bio-One 655209) in quantities of 100, 50, and 0 uL using a multi-channel pipettor (Thermo Finnpipette Model 4510 Multi-Channel Digital Pipette). 
   

• A dilution of FITC (0.0005 mg/mL in this case) was dispensed, using the VP 177AD-1, into the Black Assay Plates to make a final volume of 200 uL per well.
   

• VP 177AD-1 was washed with 0.1 M Tris-HCl, pH 8.0, three times, dispensing 200 uL into an empty Black Assay Plate after each wash.
   

• The Black Assay Plates were covered and left in the dark until ready to read.
   

•  Mixed using a Thermolyne model M16715 16700 Mixer for 5 seconds.
   

•  Read plates at A535 nm using 200 uL Tris-HCl as blank.

CONTROLS

A standard curve was generated with 2.5-fold serial dilutions of FITC in 0.1 M Tris-HCl, pH 8.0, using a multi-channel pipettor.  In this case, 10 uL of the appropriate serial dilutions of FITC was added to 190 uL Tris in an Assay Plate with a 12-channel pipettor.  The slope and y-intercept values from the linear range of the standard curve were used to determine the mean volume of FITC dispensed by the manifold.

DETERMINATION OF STANDARD CURVE DILUTION RANGE

To determine the best dilutions of FITC to use for the control standard curve, an initial test was done with two-fold serial dilutions of FITC (24 two-fold serial dilutions were used initially).  10 uL of each FITC dilution was transferred to 190 uL of Tris with a 12-channel pipettor.  This determined the best range, over 12 dilutions, to use as the control standard curve.  The appropriate dilution range for dispensing was also extrapolated from this curve. 

CONTROL STANDARD CURVE PLATE

For the control standard curve, twelve 2.5-fold serial dilutions of FITC in 0.1 M Tris-HCl, pH 8.0, were prepared and added to a row of the Control Source Plate.  The stock concentrations ranged from 0.156 to 0.000007 mg/mL in this example.  Using a 12-channel pipettor, 10 uL of each FITC dilution was added to 190 uL Tris in seven rows of the Control Assay Plate.  The last row contained 200 uL of Tris as a blank control. 

FINAL CONCENTRATION OF FITC IN ASSAY PLATES

It was determined from the Control Standard Curve Assay Plate that a final concentration between the range of 0.00008 and 0.0005 mg/mL was best in a 2.5-fold serial dilution across the 12 columns of the plate.  To test the dispenser, a single starting FITC concentration was selected such that the final FITC concentrations in the Assay Plates were between 0.0001 and 0.0005 mg/mL.

CALCULATION OF VOLUME DISPENSED

To calculate the volume of FITC transferred in each assay:

1. Read each assay plate at A535.
    

2. Calculate, and then plot, the mean values from all of the control standard curves.
    

3. Calculate the slope, y-intercept and R²-Value from the mean values in the linear range of the control
   standard curve.
    

4. Identify the A535 excitation values from the assay plate that fall within the linear range of the control standard curve (y).
    

5. Calculate, using the slope and y-intercept (m and b) from the control standard curve mean values and the A535 excitation values (y), the mg of FITC (x) per well.
    

6. Calculate the concentration of FITC in that dilution and the number of uL transferred by dispense.

  II.  REAGENTS

ORDERING AND STORAGE INFORMATION

  III.  PREPARATION OF STOCK SOLUTIONS

25 mg/mL FITC in 100% DMSO:

    Add 4 mL fresh 100% DMSO to 100 mg FITC
    Mix well
    Store @ RT, covered with foil

0.1 M Tris-HCl pH. 8.0

   Dilute 100 mL 1 M Tris in 900 mL Distilled H20.
   Store @ RT.

  IV.  PREPARATION OF FITC SERIAL DILUTIONS

A total of 350 mL of the dilution used for dispensing was used to prime the VP 177AD-1 and to fill the Assay Plates.  The dilution used was 0.00050 mg/mL FITC.  The following table was  used as a guideline for preparing the serial dilutions, including extra for the Control Standard Curve and pipetting variables. 

Note:  The FITC was first dissolved in 4 mL 100% DMSO, covered in aluminum foil, placed in a dark area, and allowed to equilibrate overnight.  All dilutions thereafter were diluted with 0.1 M Tris buffer.

Table 1
 

*Starting Dilution for Control Standard Curve
**Dilution used for dispensing

A total of 600 uL of each 2.5-fold dilution was prepared for the Control Standard Curve Source Plate.  The table below was a guideline for preparing the Control Standard Curve dilutions. 

Table 2
 

  V.  ASSAY PREPARATION

A)  Materials and Equipment
 

      One VP 177AD-1

      One VP 195D

      One 12-Channel Pipette (Thermo Finnpipette Model 4510 Multi-Channel Digital Pipette)

      Wallac Victor™ 1420 Multilabel Counter plate reader

      4 mL DMSO

      1200 mL 0.1 M Tris HCl, pH 8.0, buffer

      100 mg FITC

      20 Black Assay Plates (Greiner Bio-One 655209)

      Twelve 2 mL Centrifuge Tubes

      Four 50 mL Centrifuge Tubes

      One 85 mL Pyrex glass bottle

      One 500 mL Pyrex glass bottle

      One 1 liter Pyrex glass bottle

B)  Preparing Reagents and Plates
 

1. Prepare reagents:

1. FITC Stock Solution (25 mg/mL)

2. DMSO (4 mL)

3. 0.1 M Tris HCl, pH 8.0 ~1200 mL
    

2. Label Black Assay Plates according to the following table:

Table 3

C)  Preparing Dilutions and Standard Curve Plate
 

1. Label twelve 2.0 mL centrifuge tubes for FITC serial dilution (1-12).
    

2. Prepare FITC Dilution according to Table 1 as a guideline.
    

3. Aliquot appropriate amount of Tris dilutant to each tube according to Table 2.
    

4. Aliquot appropriate FITC dilution to first tube, and begin serial dilution from there, using Table 2 as a guideline.
    

5. Transfer 125ul of each dilution into the Standard Curve Prep Plate (#20).  Start at A1 (highest concentration) to A12 (lowest concentration), changing pipette tips after each transfer.
    

6. Use 12-channel pipette to dispense 190ul 0.1 M Tris into Standard Curve Plate (#19) and 200ul into the last row.
    

7. Transfer 10ul of each dilution from Plate #20 to each well of Plate #19 by column except for the last row.  (e.g. from Plate #20, Transfer from A1 to A1-A11 of Plate #19, B1 to B1-B11, etc.)

D)  Pre-fill Assay Plates
 

1. Use a multi-channel pipette dispenser.
    

2. Dispense 150ul 0.1M Tris to Assay Plates 1-5.
    

3. Dispense 100ul 0.1M Tris to Assay Plates 6-10.

E)    Dispense FITC to Assay Plates (See Technote 164 for guidelines to using VP 177AD-1)
 

1. Use VP 195D Bottle Top Dispenser with VP 177AD-1
    

2. Fill VP 177AD-1 with 0.0005mg/ml FITC (Priming)
    

3. Dispense 50 uL per well (4.8 mL per plate) 0.0005mg/ml FITC to Assay Plates 1-5
    

4. Dispense 100 uL per well (9.6 mL per plate) 0.0005mg/ml FITC to Assay Plates 6-10
    

5. Dispense 200 uL per well (19.2 mL per plate) 0.0005mg/ml FITC to Assay Plates 11-15
    

6. Pump excess 0.0005mg/ml FITC from VP 177AD-1

F)  Rinse Dispenser
 

1. Fill VP 177AD-1 Dispenser with 0.1M Tris, pH 8.0
    

2. Dispense 200ul 0.1M Tris to Wash Plate 1 (#16)
    

3. Pump excess Tris from Dispenser
    

4. Fill VP 177AD-1 Dispenser with fresh 0.1M Tris, pH 8.0
    

5. Dispense 200ul 0.1M Tris to Wash Plate 2 (#17)
    

6. Pump excess Tris from Dispenser
    

7. Fill VP 177AD-1 Dispenser with fresh 0.1M Tris, pH 8.0
    

8. Dispense 200ul 0.1M Tris to Wash Plate 3 (#18)
    

9. Pump excess Tris from Dispenser

G)  Reading Plates
 

1. Cover plates with adhesive plate sealers and keep in the dark until ready to be read.
    

2. Use an orbital shaker to mix plate contents.
    

3. Read each Assay Plate at A535.

  VI. Results

Aqueous Standard Curve
 

  Summary

The aqueous standard curve had a linear range between excitation signals of 1000-85000.  The mean excitation signals of the transfers fell within this range.  CV’s for the multi-channel pipettor used to generate the standard curve ranged from 4.6% to 18%.  The average CV’s for the dispense transfers ranged from 3.5% - 8.2%.  The 50 uL transfer averaged an 8.2% CV, 100 uL, 4.6%, and the 200 uL transfer, 3.5% CV.  It should be noted that CV’s were higher for the lower volume transfers, but improved with higher volume transfers.  The target 50 uL transfer dispensed an average of 49 uL, a percent difference of 2.02%.  The 100 uL transfer dispensed 101.27 uL, a 1.26% difference, and the 200 uL transfer dispensed 193.94 uL, a 3.1% difference.

  DISCUSSION

Having the excitation signals fall within the linear range of the standard curve is important because that is the readable limit of the plate reader; values outside of this range do not accurately reflect the relative concentration to excitation signal readout.  The multi-channel pipettor CV’s ranged from 4.6% to 18%; however, this range included the lower concentration transfers as well.  Excitation signal readouts of low concentrations can be inaccurate because it is more impacted by background, such as the tris buffer or even the plate itself, than higher concentrations.  Thus, a more accurate range for the CV’s of the multi-channel pipette would be in the range of 4.6% to 12.5%, where the minimum excitation signal reading is lower bound of the linear range, 1000.
 

  CONCLUSION

The VP 177AD-1 is a reliable instrument for rapid dispense of 96-well plates.  For volume transfers as little as 50 uL per well, CV’s were below 10%, a good standard for manual assay development.  For higher volume transfers, such as 100 and 200 uL per well, CV’s improved to below 5%; good enough even for automation assay development standards.  The accuracy of the device is consistent regardless of the target volume dispense and is within 3.1% of the targeted dispense volume.  The VP 177AD-1 can be interchanged with an 8 or 12-channel pipettor and still retain reliability but with increased throughput.